ABSTRACT Systemic fungal infections affect millions of people worldwide and there are limited antifungal therapeutic options. Cryptococcal meningitis is an invariably lethal fungal infection if untreated. The existing antifungal drugs are not always effective and the mortality rates of this disease are up to 70%. Finding new therapeutics and understanding their mechanisms of action is thus a critical need. Sertraline (Zoloft) is a widely prescribed drug used to treat mood disorders including depression. We discovered that sertraline (SRT) kills the deadly fungal pathogen Cryptococcus neoformans both in vitro and in animal models at clinically achievable concentrations. Furthermore, SRT and the commonly used antifungal fluconazole (FLC) act synergistically or additively against C. neoformans in vitro and in vivo. Our results showing SRT and FLC were highly effective against Cryptococcus when combined in vitro and in animal models provided the basis for a clinical trial of using SRT and FLC as a therapy for cryptococcal meningitis (ClinicalTrials.gov NCT01802385). Early results from the trial show positive indications for SRT?s clinical efficacy. A new clinical trial of adding SRT to the standard antifungal treatment against coccidioidomycosis has been launched. Although the mode of action of SRT for mood-treatment in humans is well-known, how SRT kills C. neoformans and other fungi is not known. This impedes the effort to develop novel class of antifungals or to optimize the use of SRT as an antifungal. Here we will use two unbiased approaches to determine the molecular target(s) of SRT in C. neoformans, and to understand how SRT and FLC potentiate each other?s effect. First, we will identify specific changes in transcription and translation following treatment of C. neoformans with SRT, FLC, and SRT+FLC by RNA-seq and ribosome profiling (ribo-seq). This will allow us to understand fungal responses to SRT and SRT+FLC. Second, we will determine the genes that affect cryptococcal susceptibility to SRT by screening collections of C. neoformans deletion strains. These approaches will allow us to identify promising targets/pathways to further examine the effect of disruption or overexpression of specific genes on cryptococcal susceptibility to SRT and SRT+FLC in this application. An understanding of SRT?s mechanism of action and how it synergizes with FLC are expected to guide the development of new therapeutic strategies that are more effective against Cryptococcus and potentially against other refractory fungal pathogens. Discoveries made by achieving these aims could additionally impact the development of therapies directed against other eukaryotic pathogens. For example, SRT is active against the parasite Leishmania and other protozoa. Establishing a rational basis for using SRT as an antifungal agent alone or in combinatorial therapy based on determining SRT?s targets and ultimately mechanism of action can help fill a critical gap for much-needed new anti-fungal therapeutics.